Neuromuscular diseases are characterized by impaired functioning of the muscles due to either muscle or nerve pathology (myopathies and neuropathies). The myopathies include genetic muscular dystrophies that are characterized by progressive weakness and degeneration of skeletal, heart and/or smooth muscle. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and patients often die before the age of thirty due to respiratory- or heart failure. It is caused by reading frame-shifting deletions (˜67%) or duplications (˜7%) of one or more exons, or by point mutations (˜25%) in the 2.24 Mb DMD gene, resulting in the absence of functional dystrophin. BMD is also caused by mutations in the DMD gene, but these maintain the open reading frame, yield semi-functional dystrophin proteins, and result in a typically much milder phenotype and longer lifespan. During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the transcript has emerged as a promising therapy for DMD (van Ommen et al., 2008; Yokota et al., 2007; van Deutekom et al., 2007; Goemans et al., 2011; Cirak et al., 2011). Using highly sequence-specific antisense oligonucleotides (AONs) which bind to the exon flanking or containing the mutation and which interfere with its splicing signals, the skipping of that exon can be induced during the processing of the DMD pre-mRNA. Despite the resulting truncated transcript, the open reading frame is restored and a protein is introduced which is similar to those found in BMD patients. AON-induced exon skipping provides a mutation-specific, and thus personalized, therapeutic approach for DMD patients. Several oligonucleotides are currently being developed for skipping most relevant exons of the dystrophin pre-mRNA such as exons 2, 8, 9, 17, 29, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60-63, 71-78 as described in WO 02/024906, WO2004/083446, WO2006/112705, WO2007/135105, WO 2009/139630, WO 2010/050801 or WO 2010/050802.
As the majority of the mutations cluster around exons 45 to 55, the skipping of one specific exon may be therapeutic for many patients with different mutations. The skipping of exon 51 applies to the largest subset of patients (˜13%), including those with deletions of exons 45 to 50, 48 to 50, 50, or 52. The AONs applied are chemically modified to resist endonucleases, exonucleases and RNaseH, and to promote RNA binding and duplex stability. Two different AON chemistries are currently being developed for exon 51 skipping in DMD: 2′-O-methyl phosphorothioate RNA AONs (2OMePS, GSK2402968/PRO051) and phosphorodiamidate morpholino oligomers (PMO, AVI-4658) (Goemans et al., 2011; Cirak et al., 2011). In two independent phase I/II studies, both were shown to specifically induce exon 51 skipping and at least partly restore dystrophin expression at the muscle fiber membranes after systemic administration. Although AONs are typically not well taken up by healthy muscle fibers, the dystrophin deficiency in DMD, resulting in damaged and thus more permeable fiber membranes, actually promotes uptake. In studies in the dystrophin-deficient mdx mouse model, 2′-O-methyl phosphorothioate RNA oligonucleotides have demonstrated an up to 10 times higher uptake in different muscle groups when compared to that in wild type mice (Heemskerk et al., 2010). Although the recent phase I/II results with both 2′-O-methyl phosphorothioate RNA and phosphorodiamidate morpholino AONs in DMD patients confirm this enhanced uptake in dystrophic muscle, the different chemical modifications seemed to result in a differential uptake by and distribution through muscle. The levels of novel dystrophin in both studies after 3 months of treatment were promising but still moderate and challenges the field to investigate next generation oligochemistry.
The particular characteristics of a chosen chemistry at least in part affects the delivery of an AON to the target transcript: administration route, biostability, biodistribution, intra-tissue distribution, and cellular uptake and trafficking. In addition, further optimization of oligonucleotide chemistry is conceived to enhance binding affinity and stability, enhance activity, improve safety, and/or to reduce cost of goods by reducing length or improving synthesis and/or purification procedures. Multiple chemical modifications have become generally and/or commercially available to the research community (such as 2′-O-methyl RNA and 5-substituted pyrimidines and 2,6-diaminopurines), whereas most others still present significant synthetic effort to obtain. Especially preliminary encouraging results have been obtained using 2′-O-methyl phosphorothioate RNA containing modifications on the pyrimidine and purine bases as identified herein.
In conclusion, to enhance the therapeutic applicability of AONs for DMD, there is a need for AONs with further improved characteristics.